This invention relates to protein kinase inhibitors, pharmaceutical compositions and dosage forms comprising them, and methods of their use for the treatment and prevention of diseases such as, but not limited to, cancer.
Cellular signal transduction is a mechanism whereby external stimuli that regulate cellular processes are relayed from receptors at the surface of a cell to its interior. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. The phosphorylation state of a protein, which can affect its conformation, enzymatic activity, and cellular location, is modified through the reciprocal actions of protein kinases (xe2x80x9cPKsxe2x80x9d) and protein phosphatases. The regulation, or lack of regulation, of protein kinases can thus have a dramatic effect on cellular behavior.
During cellular signal transduction, the function of each receptor kinase is determined by its pattern of expression, ligand availability, and the array of downstream signal transduction pathways that are activated by it. One example of a pathway includes a cascade of Growth Factor receptor tyrosine kinases (xe2x80x9cRTKsxe2x80x9d), such as EGF-R, PDGF-R, VEGF-R, IGF1-R, and the Insulin receptor, that deliver signals via phosphorylation to other kinases, such as Src tyrosine kinase and Raf, Mek, and Erk serine/threosine kinases. See, e.g., Davis, B. D., et al., Microbiology 838-841 (4th ed., 1990); and Brott, B. K., et al., Cell Growth Differ. 4(11):921-929 (1993). Each of these kinases play related, but functionally distinct, roles. The loss of regulation of the Growth Factor signaling pathway is a frequent occurrence in disease states such as cancer.
Aberrant expression of, or mutations in, protein kinases have been shown to lead to either uncontrolled cell proliferation (for example, malignant tumour growth) or to defects in key developmental processes. Protein kinases have been implicated as targets in central nervous system disorders (such as Alzheimer""s), inflammatory disorders (such as psoriasis), bone diseases (such as osteoporosis), atheroscieroses, restenosis, thrombosis, metabolic disorders (such as diabetes), and infectious diseases (such as viral and fungal infections).
Because the regulation and/or inhibition of protein kinases can aid in the treatment and/or prevention of a variety of diseases, significant research has been directed at discovering compounds that affect protein kinase activity. This research is similar to that which may have led to discovery of the compounds disclosed by PCT application WO 91/09598 and U.S. Pat. No. 5,811,432, which are of the formula 
wherein one of A, B, D, and E is nitrogen and the others are carbon; X and Y can be, for example, halogen or hydroxy; R1 is (C1-C6) alkyl or an amide; R2 is (C1-C8) alkyl, preferably (C3-C8)alkyl; and W is, for example, hydrogen or (C2-C10) alkanoyl. These compounds, which are not reported to be kinase inhibitors, are allegedly inhibitors of prostaglandin H2 synthase, 5-lipoxygenase, and interleukin-1 biosynthesis, and allegedly are anti-inflammatory and analgesic agents.
Examples of compounds that are allegedly protein kinase inhibitors are disclosed by PCT application WO 97/13771. These compounds are of the formula 
wherein X is nitrogen or CH; R2, Y, R3, and R5 are each selected from a large number of moieties; the group 
represents, for example, a 5-membered heterocyclic ring; and each R1 independently represents a 5- or 6-membered heterocyclic ring.
PCT application WO 98/02437 discloses compounds similar in structure to those described above, i.e.: 
As above, R1 represents a 5- or 6-membered heterocyclic ring. These compounds are also allegedly protein tyrosine kinase inhibitors.
PCT application WO 98/02438 discloses compounds of the formula: 
wherein X is nitrogen or CH; R1, R2, and Y are each selected from a large number of moieties; U is a 5- to 10-membered mono or bicyclic ring system; the group 
represents, for example, a 5-membered heterocyclic ring; and Rxe2x80x3 represents a phenyl group or a 5- or 6-membered heterocyclic ring. These compounds are also allegedly protein tyrosine kinase inhibitors.
PCT application WO 98/23613 discloses compounds of the formula: 
wherein Z can be a group of the formula 
and R6 is H, halogen, cyano, alkyl, or substituted alkyl. These compounds can allegedly be used in the treatment of hyperpoliferative diseases such as cancer.
PCT application WO 99/21859 discloses compounds of the formula: 
wherein the R groups are variously defined. These compounds are reportedly useful as protein kinase inhibitors.
PCT application WO 99/37622 discloses a compound of the formula: 
which is allegedly useful as a PDE4 and TNF-xcex1 antagonist.
U.S. Pat. No. 5,916,891 discloses a compound of the formula: 
which is a derivative of the compound of formula: 
which is allegedly a p38/Raf inhibitor. Both of these compounds share structural features with rofecoxib, which is sold by Merck under the tradename Vioxx(copyright) and which has the formula: 
and celecoxib, which is sold by Monsanto and which has the formula: 
Final examples of compounds that are allegedly useful as protein kinase inhibitors are disclosed in PCT applications WO 87/04928 and WO 96/16964.
Despite the large number of compounds that reportedly inhibit protein kinase activity, a need still exists for compounds that can be used in the treatment and/or prevention of cancer and other diseases in humans. This is due, in part, to bioavailability, toxicity, and other problems which render many of the known protein kinase inhibitors unsuited for clinical development.
This invention is therefore directed in part to compounds which modulate protein kinase (xe2x80x9cPKxe2x80x9d) signal transduction by affecting the enzymatic activity of tyrosine kinases and thereby interfering with the signals transduced by them. More particularly, the present invention is directed to compounds which modulate the RTK, cellular tyrosine kinase (xe2x80x9cCTKxe2x80x9d) and/or serine/threonine kinase (xe2x80x9cSTKxe2x80x9d) mediated signal transduction pathways as a therapeutic approach to treat many kinds of solid tumors, including but not limited to carcinoma, sarcomas including Kaposi""s sarcoma, leukemia, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma. Other specific indications related to these include, but are not limited to, brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers, blood cancers, lung cancers, bone cancers and leukemias.
Further examples, without limitation, of the types of disorders related to unregulated PK activity that the compounds described herein may be useful in preventing, treating and/or studying, are cell proliferative disorders, fibrotic disorders and metabolic disorders. Cell proliferative disorders, which may be prevented, treated or further studied by the present invention include cancers, blood vessel proliferative disorders and mesangial cell proliferative disorders.
Blood vessel proliferative disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels. The formation and spreading of blood vessels, or vasculogenesis and angiogenesis, respectively, play important roles in a variety of physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. They also play a pivotal role in cancer development. Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness. Conversely, disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis, are also implicated.
Fibrotic disorders refer to the abnormal formation of extracellular matrices. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar. Hepatic cirrhosis can cause diseases such as cirrhosis of the liver. An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis. Lipocytes appear to play a major role in hepatic cirrhosis. Other fibrotic disorders implicated include atherosclerosis. Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells. Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. The PDGF-R has been implicated in the maintenance of mesangial cell proliferation. Floege et al., Kidney International 43:47S-54S (1993).
As noted previously, PKs have been associated with such cell proliferative disorders. For example, some members of the RTK family have been associated with the development of cancer. Some of these receptors, like the EGFR (Tuzi et al., Br. J. Cancer 63:227-233 (1991); Torp et al., APMIS 100:713-719(1992)) HER2/neu (Slamon et al., Science 244:707-712 (1989)) and PDGFR (Kumabe et al., Oncogene, 7:627-633 (1992)) are over-expressed in many tumors and/or are persistently activated by autocrine loops. In fact, in the most common and severe cancers these receptor over-expressions have been demonstrated Akbasak and Suner-Akbasak et al., J Neurol. Sci., 111:119-133 (1992); Dickson et al., Cancer Treatment Res. 61:249-273 (1992); Korc et al., J. Clin. Invest. 90:1352-1360 (1992)) and autocrine loops (Lee and Donoghue, J Cell. Biol., 118:1057-1070 (1992); Korc et al., supra; Akbasak and Suner-Akbasak et al., supra). For example, EGFR has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer. PDGFR has been associated with glioblastoma, lung, ovarian, melanoma and prostate. The RTK c-met has been generally associated with hepatocarcinogenesis and thus hepatocellular carcinoma. C-met has been linked to malignant tumor formation. More specifically, the RTK c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic and gastric carcinoma, leukemia and lymphoma. Additionally, over-expression of the c-met gene has been detected in patients with Hodgkins disease, Burkitts disease, and the lymphoma cell line. Flk has been associated with a broad spectrum of tumors including without limitation mammary, ovarian and lung tumors as well as gliomas such as glioblastoma IGF-IR, in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers. For example, IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g., human breast cancer carcinoma cells (Arteaga et al., J. Clin. Invest. 84:1418-1423 (1989)) and small lung tumor cells (Macauley et al., Cancer Res., 50:2511-2517 (1990)). In addition, IGF-I, integrally involved in the normal growth and differentiation of the nervous system, appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al., Cancer Res. 53:2475-2478 (1993). The importance of the IGF-IR and its ligands in cell proliferation is further supported by the fact that many cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cells of the bone marrow) are stimulated to grow by IGF-I. Goldring and Goldring, Eukaryotic Gene Expression, 1:301-326 (1991). In a series of recent publications, Baserga even suggests that IGF-IR plays a central role in the mechanisms of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, Cancer Res., 55:249-252 (1995); Baserga, Cell, 79:927-930 (1994); Coppola et al., Mol. Cell. Biol., 14:45884595 (1994). STKs have been implicated in many types of cancer including notably breast cancer. Cance, et al., Int. J. Cancer, 54:571-77 (1993).
The association between abnormal PK activity and disease are not restricted to cancer, however. For example, RTKs have been associated with diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer""s disease, epidermal hyperproliferation and neurodegenerative diseases, age-related macular degeneration, hemangiomas. For example, EGFR is indicated in corneal and dermal wound healing. Defects in the Insulin-R and the IGF-IR have been indicated in type-II diabetes mellitus. A more complete correlation between specific RTKs and their therapeutic indications is set forth in Plowman et al., DNandP 7:334-339 (1994). As noted previously, not only RTKs but CTKs as well including, but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolen et al., FASEB J., 6:3403-3409 (1992)) are involved in the proliferative and metabolic signal transduction pathway and thus would be expected, and in fact have been shown, to be involved in many PTK-mediated disorders to which the present invention is directed. For example, mutated src (v-src) has been demonstrated as an oncoprotein (pp60v-src) in chicken. Moreover, its cellular homolog, the proto-oncogene pp60v-src transmits oncogenic signals of many receptors. For example, over-expression of EGFR or HER2/neu in tumors leads to the constitutive activation of pp60c?src, which is characteristic for the malignant cell but absent from the normal cell. On the other hand, mice deficient in the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders. Similarly, Zap70 is implicated in T-cell signaling.
PKs have been implicated in other diseases and disorders. For example, STKs have been associated with inflamation, autoimmune disease, immunoresponses, and hyperproliferation disorders such as restinosis, fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis. PKs have also been implicated in embryo implantation and the compounds of this invention may provide an effective method of preventing embryo implantation. Finally, both RTKs and CTKs are currently suspected as being involved in hyperimmune disorders.
This invention encompasses novel 4-substituted 7-aza-indolin-2-ones, pharmaceutical compositions and dosage forms comprising them, methods of their use as protein kinase inhibitors, and methods of their use for the treatment and/or prevention of disease.
A first embodiment of the invention encompasses a compound of Formula 1
or a pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein:
R1 is H or methyl;
each of R2 and R3 is independently H, halogen, (C1-C3)alkyl, or (C1-C3)alkoxy; or R2 and R3 taken together form an optionally substituted methylindene or a 3- to 7-membered ring optionally comprising 0-3 heteroatoms;
R4 is H, methyl, trifluoromethyl, (C1-C4)alkyl, alkoxy, amido, amino, or optionally substituted aryl;
X is a chemical bond, ethynyl, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O2)xe2x80x94, xe2x80x94NR5C(O)xe2x80x94, or xe2x80x94NR5xe2x80x94, wherein R5 is H, methyl, or substituted methylene;
Y is a 5- to 10-membered mono or bicyclic, saturated, unsaturated, or aromatic ring comprising 0-3 heteroatoms and optionally substituted; and
Z is N or CR6, wherein R6 is H, halogen, nitro, cyano, alkoxyl, sulfonamide, amino, or amide.
Preferred compounds of Formula 1 are those wherein X is a chemical bond, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, or xe2x80x94NR5xe2x80x94.
Additional preferred compounds of Formula 1 are those wherein Y is selected from the group consisting of phenyl, indolyl, indolinyl, 1H-indazolyl, 2,3-dihydro-1H-indazolyl, 1H-benzimidazolyl, 2,3-dihydro-1H-benzimidazolyl, benzotriazolyl, pyridyl, pyrimidyl, 4-substituted piperazin-1-yl, morpholino, piperidinyl, pyrrolidin-1-yl, furanyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, pyridopyrrolyl, pyridazopyrrolyl, pyrimidopyrrolyl, pyrazopyrrolyl, pyridofuranyl, and derivatives thereof.
Additional preferred compounds of Formula 1 are those wherein Z is N or CH.
Additional preferred compounds of Formula 1 are those wherein R2 and R3 are both H, halogen, or methyl.
Additional preferred compounds of Formula 1 are those wherein R2 and R3 are taken together to form a ring selected from the group consisting of 1,3-dioxolane, 1,3-dioxane, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
Additional preferred compounds of Formula 1 are those wherein R2 and R3 are taken together to form an optionally substituted methylindene selected from those of Formulas 1a-1n: 
wherein:
n is an integer of 0-3;
each R7 is independently H, alkyl, carboxylic acid, amine, halogen, nitro, cyano, X1, X2xe2x80x94(C1-C4)alkyl-R8, X2xe2x80x94(C1-C4)alkenyl-R8, or X2xe2x80x94(C1-C4)alkynyl-R8;
X1 is xe2x80x94C(O)NR9xe2x80x94, xe2x80x94NR9C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, C(O)R11, xe2x80x94OC(O)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NR9xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94S(O2), or xe2x80x94S(O2)NR9xe2x80x94;
X2 is a chemical bond, xe2x80x94C(O)NR9xe2x80x94, xe2x80x94NR9C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, C(O)R11, xe2x80x94OC(O)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NR9xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94S(O), or xe2x80x94S(O2)NR9xe2x80x94;
R8 is selected from the group consisting of hydrogen, dialkylamnino, carboxyl, hydoxyl, alkoxy, sulfonamide, urea, carbamate, diol, alkylsulphonyl, and R10;
R9 is H or (C1-C3)alkyl;
R10 is an optionally substituted 5- or 6-membered saturated, unsaturated, or aromatic heterocycle comprising from 1 to 4 heteroatoms; and
R11 is an optionally substituted 5- or 6-membered saturated heterocyclic ring.
In more preferred compounds of the invention, R7 is X2xe2x80x94(C1-C4)alkyl-R8, X2xe2x80x94(C1-C4)alkenyl-R3, or X2xe2x80x94(C1-C4)alkynyl-R8, and R8 is selected from the group consisting of alkylsulfonyl, alkoxy, carboxyl, morpholino, 1-alkyl-piperazin-4-yl, pyrrolidinyl, piperidinyl, pyridyl, imidazolo, triazolo, tetrazolo, and thiazolo.
Additional preferred compounds of the invention are those of Formula 1 wherein R4 is H, methyl, or trifluoromethyl.
Additional preferred compounds of the invention are those of Formula 1 wherein if Z is CH, R1 is CH3 or R3 and R2 do not form an optionally substituted methylindene.
Specific preferred compounds of the invention are those of Formulas 3-54: 
and pharmaceutically acceptable salts, solvates, clathrates, and prodrugs thereof. Preferred pharmaceutically acceptable salts of the compounds of the invention are hydrochloride salts.
A second embodiment of the invention encompasses a method of preparing a compound of Formula 2: 
wherein X, Y, Z, R1, R4, R7, and n are defined above, which comprises reacting a compound of the formula: 
wherein L is a leaving group with a compound of formula YXH under conditions sufficient to form a compound of Formula 2. Preferred leaving groups include, but are not limited to, Br, Cl, SCH3, and S(O)CH3. In a preferred method of this embodiment, the reaction is performed in a solvent. Preferred solvents are polar. More preferred solvents include, but are not limited to, alcohols, DMF, DMSO, and mixtures thereof. In another preferred method of this embodiment, the reaction is catalyzed by a catalyst such as, but are not limited to, AgOTf, Pd(Ph3)4, and p-TsOH.
A third embodiment of the invention encompasses a method of preparing a compound of Formula 2 which comprises reacting a compound of the formula: 
with a compound of the formula: 
under conditions sufficient to form a compound of Formula 2. In a preferred method of this embodiment, the reaction is performed in a solvent. Preferred solvents are polar. More preferred solvents include, but are not limited to, alcohols, DMF, DMSO, and mixtures thereof. In another preferred method of this embodiment, the reaction is catalyzed by a base. Preferred bases include, but are not limited to, pyridine and piperidine.
A fourth embodiment of the invention encompasses a pharmaceutical composition comprising a compound of Formula 1, or a pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, and a pharmaceutically acceptable carrier. This embodiment further encompasses dosage forms suitable for oral, transdermal, topical, parenteral (e.g., subcutaneous, intrathecal, intramuscular, and intravenous), or mucosal (e.g., rectal, vaginal, and nasal) administration. Therefore, solid, lyophilized, injectable and transdermal topical formulations are contemplated herein.
A fifth embodiment of the invention encompasses a method of regulating, modulating, or inhibiting protein kinase activity which comprises contacting a compound of Formula 1, or a pharmaceutically acceptable salt or solvate thereof, with a protein kinase. In a preferred method of this embodiment, the protein kinase is a protein tyrosine kinase. Preferably the contact is made in cell culture (in vitro) or in a human, animal or bird (in vivo).
In another preferred method of this embodiment, the protein kinase is selected from the group consisting of ab1, ATK, bcr-ab1, Blk, Brk, Btk, c-fms, c-kit, c-met, c-src, CDK1, CDK2, CDK4, CDK6, cRaf1, CSF1R, CSK, EGFR, ErbB2, ErbB3, ErbB4, ERK, Fak, fes, FGFR1, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, FLK-4, flt-1, Fps, Frk, Fyn, GSK, Gst-Flk1, Hck, Her-2, Her-4, IGF-1R, INS-R, Jak, JNK, KDR,Lck, Lyn, MEK, p38, PANHER, PDGFR, PLK, PKC, PYK2, Raf, Rho, ros, SRC, tie1, tie2, TRK, UL97, VEGFR, Yes, and Zap70.
In a more preferred method of this embodiment, the protein kinase is selected from the group consisting of PANHER, EGFR, Her-2, Her-4, PDGFR, SRC, Lck, cdk2, p38, Raf, and Rho.
In an even more preferred method of this embodiment, the protein kinase is selected from the group consisting of PANHER, CDK2, PDGFR, p38, and Raf.
A sixth embodiment of the invention encompasses a method of treating or preventing a disease characterized by unregulated protein kinase activity which comprises administering to a patient (e.g., a mammal, preferably a human) in need of such treatment or prevention a therapeutically or prophylactically effective amount of a compound of Formula 1, or a pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof.
In a preferred method of this embodiment, the disease characterized by unregulated protein kinase activity is selected from the group consisting of: blood vessel proliferative disorders such as, but not limited to, arthritis and restenosis; fibrotic disorders such as, but not limited to, hepatic cirrhosis and atherosclerosis; mesangial cell proliferative disorders such as, but not limited to, glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, organ transplant rejection, and glomerulopathies; metabolic disorders such as, but not limited to, psoriasis, diabetes mellitus, chronic wounds, inflammation, and neurodegenerative diseases; auto-immune diseases; allergies; asthma; thrombosis; nervous system diseases; and cancer.
In a more preferred method of this embodiment, the disease characterized by unregulated protein kinase activity is cancer. Examples of cancers include, but are not limited to, breast, stomach, ovary, colon, lung (including non-small cell), brain, larynx, lymphatic system, genitourinary tract (including bladder and prostate), ovarian, gastric, bone, and pancreatic cancer.
As used herein, the term xe2x80x9chalogenxe2x80x9d includes fluorine, chlorine, bromine, and iodine.
As used herein, the term xe2x80x9calkylxe2x80x9d includes saturated monovalent hydrocarbon radicals having straight, cyclic, or branched moieties, and combinations thereof.
As used herein, the term xe2x80x9calkenylxe2x80x9d includes monovalent hydrocarbon radicals having straight, cyclic, or branched moieties, and combinations thereof which comprise at least one carbon-carbon double bond.
As used herein, the term xe2x80x9calkynylxe2x80x9d includes saturated monovalent hydrocarbon radicals having straight, cyclic, or branched moieties, and combinations thereof which comprise at least one carbon-carbon triple bond.
As used herein to describe a compound or moiety, the term xe2x80x9cderivativexe2x80x9d means a compound or moiety wherein the degree of saturation of at least one bond has been changed (e.g., a single bond has been changed to a double or triple bond) or wherein at least one hydrogen atom is replaced with a different atom or a chemical moiety. Examples of different atoms and chemical moieties include, but are not limited to, halogen, oxygen, nitrogen, sulfur, hydroxy, methoxy, alkyl, amine, amide, ketone, and aldehyde.
As used herein to describe a compound or moiety, the term xe2x80x9csubstitutedxe2x80x9d means a compound or moiety wherein at least one hydrogen atom is replaced with a different atom or a chemical moiety. Examples of different atoms and chemical moieties include, but are not limited to, halogen, oxygen, nitrogen, sulfur, hydroxy, methoxy, alkyl, amine, amide, ketone, and aldehyde.
As used herein, the term xe2x80x9cheteroatomxe2x80x9d means an atom selected from the group consisting of O, S, and N.
As used herein, the term xe2x80x9cmethylindenexe2x80x9d means an optionally substituted carbon-carbon double bond.
As used herein, the term xe2x80x9cprodrugxe2x80x9d means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide the compound. Examples of prodrugs include, but are not limited to, derivatives of compounds of Formula 1 that comprise biohydrolyzable moieties such as amides, esters, carbamates, carbonates, or ureides. Such biohydrolyzable moieties may be linked, for example, to a peptide.
As used herein, the terms xe2x80x9cbiohydrolyzable carbamate,xe2x80x9d xe2x80x9cbiohydrolyzable carbonate,xe2x80x9d and xe2x80x9cbiohydrolyzable ureidexe2x80x9d mean a carbamate, carbonate, or ureide, respectively, of a compound that either: 1) does not interfere with the biological activity of the compound but can confer upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) is biologically inactive but is converted in vivo to the biologically active compound. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines.
As used herein, the term xe2x80x9cbiohydrolyzable esterxe2x80x9d means an ester of a compound that either: 1) does not interfere with the biological activity of the compound but can confer upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) is biologically inactive but is converted in vivo to the biologically active compound. Examples of biohydrolyzable esters include, but are not limited to, lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters.
As used herein, the term xe2x80x9cbiohydrolyzable amidexe2x80x9d means an amide of a compound that either: 1) does not interfere with the biological activity of the compound but can confer upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) is biologically inactive but is converted in vivo to the biologically active compound. Examples of biohydrolyzable amides include, but are not limited to, lower alkyl amides, xcex1-amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
As used herein, the term xe2x80x9cdisease characterized by unregulated protein kinase activityxe2x80x9d means a disease or condition caused or aggravated by abnormal kinase activity. Examples of such diseases or conditions include, but are not limited to, uncontrolled cell proliferation (e.g., malignant tumour growth) or to defects in key developmental processes. Specific examples include, but are not limited to, central nervous system disorders, inflammatory disorders, bone diseases, atheroscieroses, restenosis, thrombosis, metabolic disorders, and infectious diseases.
As used herein, the term xe2x80x9ctreatxe2x80x9d includes the amelioration, reduction, or eradication of the symptoms of disease.
This invention encompasses compounds that affect the activity of one or more protein kinases. The invention further encompasses compounds that are useful in methods of regulating, modulating, and/or inhibiting protein kinases of both the receptor and non-receptor types. The invention further provides methods of treating and preventing diseases and disorders that are related to unregulated protein kinase activity in birds and animals, and particularly in mammals such as humans.
Compounds of the invention are those of Formula 1
and pharmaceutically acceptable salts, solvates, clathrates, and prodrugs thereof, wherein R1, R2, R3, R4, X, Y, and Z are defined herein. It should be appreciated that certain compounds of the invention may have one or more chiral centers or axes, thus the invention further encompasses racemic and optically pure enantiomers of compounds of Formula 1. The invention also encompasses crystalline and amorphous forms as well as lyophilized, non-lyophilized, and sterile compositions of compounds of Formula 1.
Pharmaceutically acceptable salts of compounds of Formula 1 include salts of acidic or basic moieties. The compounds of the invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts that contain pharmacologically acceptable anions such as, but not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate [i.e., 1,1xe2x80x2-methylene-bis-(2-hydroxy-3-naphthoate)]. Compounds of the invention that comprise a basic moiety, such as an amino group, can form pharmaceutically acceptable salts with various amino acids in addition to the acids mentioned above.
Compounds of the invention that are acidic in nature are capable of forming base addition salts with various pharmacologically acceptable cations. Examples of such salts include, but are not limited to, the alkali metal or alkaline earth metal salts, e.g., calcium, magnesium, sodium, and potassium salts.
Compounds of the invention can be readily prepared from 4-chloro-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one or 4-chloro-5,7-dihydro-pyrrolo[2,3-d]pyrimidine-6-one or other commercially or readily accessible starting material. A preferred method of preparing 4-chloro-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one is shown in Scheme 1: 
According to Scheme 1, compound (a) is chlorinated to provide compound (b). Although a variety of reaction conditions known to those skilled in the art can be used to chlorinate compound (a), the use of POCl3 is preferred. Compound (b) is then converted to compound (c) using C5H5Nxe2x80x94HBrxe2x80x94Br2 in t-butanol. This reaction is preferably run at room temperature for about four hours, although those skilled in the art will recognize that the solvent and the reaction temperature and time can be varied to maximize the yield of compound (c). Finally, 4-chloro-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one (compound (d)) is formed by replacing the bromine atoms bound to compound (c) with hydrogen atoms. This is readily accomplished using zinc in acetic acid, although other methods known to those skilled in the art can also be used.
A preferred method of preparing 4-chloro-5,7-dihydro-pyrrolo[2,3-d]pyrimidine-6-one is described in Scheme 2: 
According to Scheme 2, compound (e) is reacted with formamidine hydrochloride under basic conditions to provide compound (f). Compound (f) is then cyclized under acidic conditions, preferably using 1N aqueous HCl, to provide compound (g). The keto moiety of compound (g) is next replaced with a chlorine using, for example, POCl3. The resulting compound (h) is then converted to compound (i) using C5H5Nxe2x80x94HBrxe2x80x94Br, in t-butanol. This reaction is preferably run at room temperature for about four hours, although those skilled in the art will recognize that the solvent and the reaction temperature and time can be varied to maximize the yield of compound (i). Finally, 4-chloro-5,7-dihydro-pyrrolo[2,3-d]pyrimidine-6-one (compound (j)) is formed by replacing the bromine atoms bound to compound (i) with hydrogen atoms. This is readily accomplished using zinc in acetic acid, although other methods known to those skilled in the art can also be used.
Compounds of the invention are readily prepared from 4-chloro-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one and 4-chloro-5,7-dihydro-pyrrolo[2,3-d]pyrimidine-6-one. A preferred method is shown in Scheme 3, wherein each of X, Y, Z, R1, R4, R7, L, and n are defined herein: 
According to Scheme 3, the starting material (k) is coupled with a derivative of pyrrole under suitable reaction conditions to yield compound (1). The leaving group L is then replaced with the moiety XY to provide compound 2.
Another preferred method of preparing compounds of the invention is shown in Scheme 4: 
According to Scheme 4, compound (m) is coupled with XY to form compound (n). Compound (n) is then converted to compound (o) using C5H5Nxe2x80x94HBrxe2x80x94Br2 in t-butanol. This reaction is preferably run at room temperature for about four hours, although those skilled in the art will recognize that the solvent and the reaction temperature and time can be varied to maximize the yield of compound (o). Finally, compound (o) is coupled with a derivative of pyrrole under suitable reaction conditions to yield compound 2.
The ability of a compound of the invention to affect the activity of a protein kinase can be readily determined using methods well known to those skilled in the art. For example, a compound can be contacted (in vitro or in vivo) with cells that express a kinase of interest, after which: (a) phenotypic changes in the cell culture can be scored as compared to control cells that were not exposed to the compound; or (b) cell lysates can be prepared to assess phosphorylated proteins.
This latter approach is illustrated by several methodologies. A common technique involves incubating cells with a ligand and radioactive phosphate, lysing the cells, separating cellular components using an SDS-polyacrylamide gel (SDS-PAGE) technique, in either one or two dimensions, and then detecting the presence of phosphorylated proteins by exposing X-ray film. A similar technique involves separating cellular components using SDS-PAGE, transferring the separated components to a solid support such as a sheet of nitrocellulose, and then detecting the presence of phosphorylated tyrosines using an antiphosphotyrosine antibody (anti-PY). The anti-PY can be detected by labeling it with a radioactive substance, which then requires scanning the labeled nitrocellulose with a piece of specialized equipment designed to detect radioactivity or exposure of X-ray film. Alternatively, the anti-PY can be labeled with an enzyme, such as horseradish peroxidase, and detected by subsequent addition of a colourometric substrate for the enzyme. A further alternative involves detecting the anti-PY by reacting it with a second antibody that recognizes the anti-PY and is labeled with either a radioactive moiety or an enzyme as previously described. Examples of these and similar techniques are described in Hansen et al., Electrophoresis 14:112-126 (1993); Campbell et al., J. Biol. Chem. 268:7427-7434 (1993); Donato et al., Cell Growth and Diff. 3:258-268 (1992); and Katagiri et al., J. Immunol. 150:585-593 (1993).
Other, ELISA-type, assays that can be used to determine the biological activity of a compound of the invention are disclosed by Peraldi et al., J Biochem. 285:71-78 (1992); Schraag et al., Analytical Biochemistry 211:233-239 (1993); Cleavland, Analytical Biochemistry 190:249-253 (1990); Farley, Analytical Biochemistry 203:151-157 (1992); and Lazaro, Analytical Biochemistry 192:257-261 (1991).
A preferred method of determining the ability of a compound of the invention to affect protein kinase activity is disclosed by U.S. patent application Ser. No. 08/234,440, which is incorporated herein by reference. According to this method, a target cell that expresses a kinase and is phosphorylated or dephosphorylated during signal transduction is exposed to a compound of the invention. The target cell is thereafter lysed to release cellular contents, which include the protein substrate. The substrate is isolated by contacting the cell lysate with a substrate-specific antibody immobilized on a solid support and subsequently washing away other cellular components. An immunoassay is performed on the isolated substrate to detect the presence or absence of phosphotyrosine residues on the substrate as compared to lysates of control target cells that were not exposed to the compound of interest. Other preferred methods of measuring the biological effects of compounds of the invention are described below in Examples 20-24.
Compounds of the invention (herein also referred to as xe2x80x9cactive ingredientsxe2x80x9d or xe2x80x9cactive compoundsxe2x80x9d) can be used to regulate, modulate, or inhibit protein kinase activity. They can thus be used in the treatment and/or prevention of a disease or disorder in birds and animals. Preferred patients are mammals, and particularly humans. Examples of diseases and disorders that can be treated or prevented by methods of the invention include, but are not limited to, blood vessel proliferative disorders such as, but not limited to, arthritis and restenosis; fibrotic disorders such as, but not limited to, hepatic cirrhosis and atherosclerosis; mesangial cell proliferative disorders such as, but not limited to, glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, organ transplant rejection, and glomerulopathies; metabolic disorders such as, but not limited to, psoriasis, diabetes mellitus, chronic wounds, inflammation, and neurodegenerative diseases; auto-immune diseases; allergies; asthma; thrombosis; nervous system diseases; and cancer. Examples of cancers include, but are not limited to, breast, stomach, ovary, colon, lung (including non-small cell lung cancer), brain, larynx, lymphatic system, genitourinary tract (including bladder and prostate), ovarian, gastric, bone, and pancreatic cancer.
4.3.1. Routes of Administration and Dosage Forms
Compounds of the invention can be administered to a patient by any suitable route, including, but not limited to, oral, transdermal, topical, parenteral (e.g., subcutaneous, intrathecal, intramuscular, and intravenous), and mucosal (e.g., rectal, vaginal, and nasal) routes. Dosage forms encompassed by the invention include, but are not limited to, tablets, caplets, capsules, troches, dispersions, suspensions, suppositories, solutions, creams, patches, solutions, lyophilized solids suitable for reconstitution into solutions, and aerosols (e.g., in the form of minipumps).
A compound of the invention can be administered in a local rather than systemic manner by, for example, injection of the compound directly into a solid tumor, often in a depot or sustained release formulation. A compound can further be administered in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposome will be targeted to, and taken up selectively by, the tumor.
Pharmaceutical compositions of the invention can be manufactured in a manner well known to those skilled in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions can thus be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into pharmaceutically acceptable preparations. Proper formulation is dependent upon the route of administration chosen.
For injection, an active ingredient can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks""s solution, Ringer""s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, an active ingredient can be combined with one or more of the many pharmaceutically acceptable carriers well known in the art. Such carriers enable compounds of the invention to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions for oral ingestion by a patient. Pharmaceutical preparations for oral use can be obtained by admixing a compound of the invention with a solid excipient, optionally grinding the resulting mixture, processing the mixture into granules, and adding optional suitable auxiliaries to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores, which can be lactose-free, are provided with suitable coatings to provide tablets of the invention. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations that can be orally administered include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain an active ingredient in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compound can be dissolved or suspended in any suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers can also be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, dosage forms of the invention can take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, active ingredients of the invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.
Compounds of the invention can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Active ingredients of the invention can also be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
Compounds of the invention can also be formulated in rectal compositions such as suppositories or retention enemas containing, for example, conventional suppository bases such as cocoa butter or other glycerides. The compounds disclosed herein can further be formulated as depot preparations. Such long acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, an active ingredient can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as a sparingly soluble derivative, for example as a sparingly soluble salt.
A preferred pharmaceutical carrier for compounds of the invention that are hydrophobic is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system can be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system can be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components can be varied: for example, other low-toxicity nonpolar surfactants can be used instead of polysorbate 80; the fraction size of polyethylene glycol can be varied; other biocompatible polymers can replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides can substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity. Additionally, compounds of the invention can be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. A variety of sustained-release materials are well known by those skilled in the art. Sustained-release capsules can, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization can be employed.
Pharmaceutical compositions also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
In addition to the common dosage forms set out herein, the compounds of the invention can be administered by controlled release means and/or delivery devices including, but not limited to, Alzet(copyright) osmotic pumps which are available from Alza Corporation, Palo Alto, Calif. Suitable delivery devices are described in U.S. Pat. Nos. 3,536,809; 3,598,123; 3,845,770; 3,916,899; 3,944,064; and 4,008,719, the disclosures of which are incorporated herein by reference.
Compositions of the invention can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient. The pack can, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label can include treatment of a tumor, such as a glioma or glioblastoma, and inhibition of angiogenesis.
Pharmaceutical compositions suitable for use in this invention include compositions wherein the active ingredient(s) is contained in an effective amount to achieve its intended purpose. Determination of effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
4.3.2. Dosages
For any compound used in the methods of the invention, a therapeutically or prophylactically effective dose can be initially estimated from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (ie., the concentration of the test compound which achieves a half-maximal inhibition of the kinase activity). Such information can be used to more accurately determine useful doses in humans.
A therapeutically effective amount refers to that amount of a compound that results in amelioration of symptoms or a prolongation of survival in a patient. A prophylactically effective amount refers to that amount of a compound which is sufficient to prevent or slow the onset of a disease or condition. Toxicity and therapeutic efficacy of compounds of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals wherein the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) is determined. The dose ratio between toxic and therapeutic or prophylactic effects is the therapeutic index and it can be expressed as the ratio of LD50 to ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient""s condition. See, e.g., Fingl et al., The Pharmacological Basis of Therapeutics 1 (1975).
Dosage amount and interval can be adjusted individually to provide plasma levels of the active ingredient that are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound, but can be estimated from in vitro data; e.g., the concentration necessary to achieve a 50-90% inhibition of the kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration, but high performance liquid chromatography (HPLC) assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using the MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
Usual patient dosages for systemic administration range from 1 to 2000 mg/day, commonly from 1 to 250 mg/day, and typically from 10 to 150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02 to 25 mg/kg/day, commonly from 0.02 to 3 mg/kg/day, typically from 0.2 to 1.5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5 to 1200 mg/m2/day, commonly from 0.5 to 150 mg/m2/day, typically from 5 to 100 mg/m2/day. Usual average plasma levels should be maintained within 50 to 5000 xcexcg/ml, commonly 50 to 1000 xcexcg/ml, and typically 100 to 500 xcexcg/ml, although in cases of local administration or selective uptake the effective local concentration of the drug can not be related to plasma concentration. As those skilled in the art will recognized, however, these dosages and plasma levels will vary with the patient, the disease treated (e.g., different cancers may require different dosages), the route of administration, and the particular active ingredient used. The dose, and perhaps the dosage frequency, will also vary according to the age, body weight, and response of the individual patient. It is thus recommended that infants, children, and patients over 65 years, and those with impaired renal, or hepatic function, initially receive low doses, and that they be titrated based on individual clinical response(s) and blood level(s).
Desirable blood levels can be maintained by a continuous infusion of the compound as ascertained by plasma levels measured by HPLC. It should be noted that the attending physician would know how and when to terminate, interrupt, or adjust therapy to lower dosage due to toxicity, or to bone marrow, liver, or kidney dysfunction. The attending physician would also know to adjust treatment to higher levels if the clinical response is not adequate (precluding toxicity).